DESCRIPTION: Previous work, largely from the PIs laboratory, is provided a broad picture of the evolutionary history of eukarytoes. While most biologists focus on the "crown" eukaryotes (the multicellular eukaryotes), the bulk of evolutionary history of the eukarytoic lineages resides in a vast number of understudied groups lower in the tree. In particular, the earliest known branching lineages of the eukarytoes are amitochondriate, and very little is known about the genomic structures of these lineages. To resolve some of these issues, the PI proposes three specific aims: (1) to obtain a large number of coding sequences from the basal eukaryotes using a coding-sequence enrichment strategy based on A-T composition, (2) to use 16S ribosomal sequences to further resolve the structure of the deeper branching eukaryotes, and (3) to use large ribosomal sequences to resolve the branching order of the crown radiation. Sequences from the lowest branching eukaryotes have been very difficult to obtain by standard homology-based approaches given the extreme divergence of these lineages. The PI's laboratory noted that the microsporita Spraguea lophii has a very small genome and very high A-T content, so that restriction enzymes requiring at least 2 G/C base pairs are expected to preferentially cut coding sequencing. Sequences of random clones isolated this way show that 40/100 such clones displayed highly significant similarities to genes in later-diverging eukaryotes. The PI proposes to use this approach to sequence 200-1000 random clones from S. lophii. Preliminary data suggests that at lest 400 clones can be screened before duplicate hits appear. Such detected genes will serve as homologous probes for determination of complete sequences, with 200-500 bp of flanking sequences included. This same coding sequence enrichment strategy will be applied with two other, deeply branching amitochondriates, Encephalitozoon intesinalis and Giardia lamblia. The PI also proposes to obtain small ribosomal sequences from key lineages in the lower eukaryotes and to use large ribosomal sequences to resolve the radiation in the crown eukaryotes.